Cell barcoding in microfluidics

Patent metadata

Field	Value
Publication number	US-10745741-B2
Application number	US-201615579834-A
Country	US
Kind code	B2
Filing date	Jun 27, 2016
Priority date	Jun 26, 2015
Publication date	Aug 18, 2020
Grant date	Aug 18, 2020

Abstract

The present invention relates to the field of microfluidics and in particular to methods for co-localizing a particle comprising DNA and/or RNA with a known barcode oligonucleotide. Thereby, the transcriptome or DNA of a cell can be barcoded and correlated to a cell phenotype or examined for the effect of a drug on the cell. The invention also provides microfluidic devices and systems having properties which make them particularly suitable for use in the methods of the invention.

First claim

The invention claimed is:

1. A method for co-localizing a particle comprising DNA and/or RNA with a known barcode oligonucleotide or set of components thereof in a microfluidic droplet in a microfluidics system, comprising:
(i) feeding a particle comprising DNA and/or RNA into a co-localizing channel,
(ii) passing the particle past a series of at least two valve-operated oligonucleotide inlets or sets of valve-operated oligonucleotide inlets, wherein one of the at least two oligonucleotide inlets or sets of oligonucleotide inlets is open while the particle passes it, and wherein each oligonucleotide inlet, when open, feeds a known barcode oligonucleotide or set of components thereof into the co-localizing channel, or each set of oligonucleotide inlets, when open, feeds a set of components of a known barcode oligonucleotide into the co-localizing channel, and
(iii) closing the oligonucleotide inlet or set of oligonucleotide inlets that is open after the particle has passed it and opening a different oligonucleotide inlet or set of oligonucleotide inlets of the at least two valve-operated oligonucleotide inlets or sets of valve-operated oligonucleotide inlets,

wherein
(A) the particle of step (i) and the known barcode oligonucleotide or the set of components thereof of step (ii) are fed into the co-localizing channel while not comprised in microfluidic droplets, and the method further comprises generating microfludic droplets downstream of the at least two valve-operated oligonucleotide inlets or sets of oligonucleotide inlets prior, during or after step (iii), including a microfluidic droplet comprising the particle and the known barcode oligonucleotide or components thereof, or
(B) the particle of step (i) and the known barcode oligonucleotide or the set of components thereof of step (ii) are fed into the co-localizing channel comprised in microfluidic droplets and these microfluidic droplets are fused.

2. The method of claim 1 , comprising repeating all steps one or more times with a further particle after step (i) is carried out for the particle fed into the co-localizing channel prior to this further particle, wherein for each particle passing the series of at least two valve-operated oligonucleotide inlets or sets of valve-operated oligonucleotide inlets in step (ii), a different valve-operated oligonucleotide inlet or set of valve-operated oligonucleotide inlets is open, and wherein the barcode oligonucleotide or set of components thereof fed into the co-localizing channel while the particle passes the series is predetermined or recorded.

3. The method of claim 1 , further comprise detecting the particle at a detection point in the co-localizing channel.

4. The method of claim 3 , wherein the particle is a cell and the cell is phenotyped at the detection point.

5. The method of claim 1 , comprising feeding a drug into the co-localizing channel (a) via the oligonucleotide inlet or at least one oligonucleotide inlet of the set of oligonucleotide inlets together with the barcode oligonucleotide or components thereof, or (b) via one of at least two drug inlets which opens and closes together with the oligonucleotide inlet or set of oligonucleotide inlets and is located adjacent to the oligonucleotide inlet or set of oligonucleotide inlets or within the set of oligonucleotide inlets; or opens when the particle passes the drug inlet and closes once it has passed it.

6. The method of any claim 1 , wherein the known barcode oligonucleotide is generated in the microfluidics system and generating the known barcode oligonucleotide comprises:
I. (a) feeding a first oligonucleotide into an oligonucleotide channel terminating at a oligonucleotide inlet of a set of the at least two sets of oligonucleotide inlets, wherein the first oligonucleotide comprises a priming region or alternatively inverted repeats of transposable elements, a variable region and a single-stranded annealing region,
(b) optionally feeding one or more sequential internal oligonucleotides into a further oligonucleotide channel, each oligonucleotide channel terminating at a further oligonucleotide inlet of the set of (a), wherein each sequential internal oligonucleotide comprises a variable region between two single-stranded annealing regions,
(c) feeding a terminal oligonucleotide into a further oligonucleotide channel terminating at a further oligonucleotide inlet of the set of (a), wherein the terminal oligonucleotide comprises a single-stranded annealing region, a variable region and optionally a priming region or alternatively inverted repeats of transposable elements,
wherein the single-stranded annealing regions are overlapping complementary sequences enabling the annealing of the first oligonucleotide, the optional one or more sequential internal oligonucleotides, and the terminal oligonucleotide in this order into a single linear oligonucleotide,
(d) annealing the first oligonucleotide, the optional one or more sequential internal oligonucleotides, and the terminal oligonucleotide together in this order into a single linear and at least partially double-stranded oligonucleotide via the overlapping annealing regions downstream of the oligonucleotide inlets,
(e) optionally, if the single linear oligonucleotide is partially double-stranded, extending double-stranded portions of the partially double-stranded oligonucleotide and/or filling gaps in the partially double-stranded oligonucleotide enzymatically, and
(f) ligating the annealed first oligonucleotide, the optional one or more sequential internal oligonucleotides, and the terminal oligonucleotide, or, if the first oligonucleotide is annealed directly to the terminal nucleotide, extending double-stranded portions of the resulting partially double-stranded oligonucleotide; or

II. (a) feeding a first oligonucleotide into an oligonucleotide combination channel terminating at one of the at least two valve-operated oligonucleotide inlets, wherein the first oligonucleotide comprises a priming region or alternatively inverted repeats of transposable elements, a variable region and a single-stranded annealing region,
(b) optionally feeding one or more sequential internal oligonucleotides into the oligonucleotide combination channel, wherein each sequential internal oligonucleotide comprises a variable region between two single-stranded annealing regions,
(c) feeding a terminal oligonucleotide into the oligonucleotide combination channel, wherein the terminal oligonucleotide comprises a single-stranded annealing region, a variable region and optionally a priming region or alternatively inverted repeats of transposable elements, wherein the single-stranded annealing regions are overlapping complementary sequences enabling the linking of the first oligonucleotide, the optional one or more sequential internal oligonucleotides, and the terminal oligonucleotide in this order into a single linear oligonucleotide, and
(d) annealing the first oligonucleotide, the optional one or more sequential internal oligonucleotides, and the terminal oligonucleotide together in this order into a single linear and at least partially double-stranded oligonucleotide via the overlapping annealing regions downstream of the oligonucleotide inlets, and
(e) optionally, if the single linear oligonucleotide is partially double-stranded, extending double-stranded portions of the partially double-stranded oligonucleotide and/or filling gaps in the partially double-stranded oligonucleotide enzymatically, and
(f) ligating the annealed first oligonucleotide, the optional one or more sequential internal oligonucleotides, and the terminal oligonucleotide, or, if the first oligonucleotide is annealed directly to the terminal nucleotide, extending double-stranded portions of the resulting partially double-stranded o

Assignees

• European Molecular Biology Laboratory

Inventors

• Utharala Ramesh
• Chaipan Chawaree
• Nögel Lukas
• Merten Christoph A

Classifications

• B01L2400/0424

  Dielectrophoretic forces · CPC title

• B01L2400/0439

  ultrasonic vibrations, vibrating piezo elements · CPC title

• B01L7/52

  with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples · CPC title

• B01L2300/021

  Identification, e.g. bar codes · CPC title

• B01L2200/141

  Preventing contamination, tampering · CPC title